The long-term objective of this research is an elucidation of aspects of the structure and structure-function relationships of rabbit muscle phosphofructokinase. The specific aims of this application are a) to affinity label the substrate and allosteric modifier binding sites on phosphofructokinase, b) to determine the effect of these specific modifications on the molecular, regulatory and kinetic properties of the enzyme as a function of the extent of modification, c) to subject the various affinity labeled phosphofructokinase preparations to a limited tryptic digestion in order to cleave each phosphofructokinase monomer into two "half-molecules" and then to determine which "half-molecule" contains each specific affinity label and d) to further degrade by chemical and enzymatic cleavages each affinity labeled "half-molecule" into smaller peptides which will be sequenced to determine which amino acid is labeled as well as the primary structure of the segment of the polypeptide chain that contributes this amino acid residue to that specific binding site. Completion of these studies should contribute to our understanding of the structure-function relationships and the molecular basis for the complex regulatory properties of this enzyme. This should contribute to our knowledge of enzyme regulatory mechanisms in general, an area of considerable medical importance since these control mechanisms are responsible for the rapid adjustment and coordination of the rates of metabolic pathways in all living organisms.